A new real-time PCR method for identification of two pathogenic Eimeria species in sheep, Eimeria ovinoidalis and Eimeria crandallis
Résumé
Coccidiosis in lambs, caused by intestinal parasites of the Eimeria genus, has high medical and economic impacts throughout the world. Diagnosis of lamb coccidiosis is currently performed in laboratory by microscopic examinations of fecal material but this method is time-consuming. Moreover, the species identification, based on microscopy, requires experienced personnel for accurate identification as oocysts of many different species are difficult to distinguish morphologically. As an alternative to morphological approach, a real-time Polymerase Chain Reaction using Taqman probes has been developed to quantify all Eimeria oocysts in the sample and to identify and quantify more specifically oocysts of Eimeria ovinoidalis and Eimeria crandallis, the species associated with clinical ovine coccidiosis. As available genomic data are scarce, a strategy of sequencing was performed on different Eimeria spp oocysts, isolated by micromanipulation. Sequences of 18S were used to designing primers and probes for three PCR test: a Pan-Eimeria test for estimate the total number of oocysts, a Eimeria crandallis test and a Eimeria ovinoidalis test for the amount of oocysts of each pathogenic species present in a fecal sample. The Locked Nucleic Acid technology was used to increase the specificity of the PCR reactions. Thanks to a modified pUC57 plasmid from Escherichia coli and an estimation of the average of gene’s copy number within and between species, the standard curve accurately quantifies all oocysts in the sample and the proportion of each pathogenic species. This new diagnostic tool could be used in sheep farms for monitoring ovine coccidiosis and for an early detection of pathogenic species.
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